1. Evaluation Of A New Algorithm In Classification Of Primary Systemic Vasculitis 2. IgG Subclass Distribution, Affinity And Epitope Analysis Of Anti-myeloperoxidase (MPO) Antibodies In Sera From Patients With Wegener's Granulomatosis And Microscopic Po

Part one: Evaluation of a new algorithm in classification of primary systemic vasculitisObjective: A new consensus algorithm for classification of anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) and polyarteritis nodosa (PAN) had been proposed by Watt et al. for epidemiological studies. In order to evaluate this algorithm, the current study used the algorithm to reclassify the patients with AAV and PAN in our center.Methods: 550 Chinese patients with primary systemic vasculitis diagnosed in our referral diagnostic center during the past ten years were retrospectively studied. 493 out of 550 were ANCA positive. We compared the new consensus algorithm and the 1994 Chapel Hill Consensus Conference (CHCC) definitions supplemented with surrogate parameters, in the same cohort of patients with primary systemic vasculitis.Results: Applying the CHCC definitions with surrogate parameters, the diagnoses were Churg-Strauss syndrome (CSS) (n=0), Wegener's granulomatosis (WG) (n=127), microscopic polyangiitis (MPA) (n=363), polyarteritis nodosa (PAN) (n=4) and unclassified (n=56), respectively. Using the new consensus algorithm, the diagnoses were CSS (n=2), WG (n=199), MPA (n=329), PAN (n=0) and unclassified (n=20), respectively.Conclusions: Watts'algorithm was a useful method to classify patients into a single category, with less unclassified patients and without overlapping diagnosis, which allow their epidemiology application. PART TWO: IgG subclass distribution, affinity and epitope analysis of anti-myeloperoxidase (MPO) antibodies in sera from patients with Wegener's granulomatosis and microscopic polyangiitisObjective: Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) were serologic marker for Wegener's granulomatosis (WG), while ANCA against myeloperoxidase (MPO) were serologic marker for microscopic polyangiitis (MPA). However, our previous study suggested that patients with MPO-ANCA positive WG were common in Chinese, indicating that patients with positive serum MPO-ANCA could manifest as either WG or MPA. The aim of this study was to compare the immunological characteristics of MPO-ANCA in patients with WG and MPA.Methods: Fifteen patients with WG and twenty one patients with MPA were enrolled in the current study. Anti-MPO IgG subclasses and their titers were detected by antigen specific ELISAs using specific monoclonal antibodies as second antibodies. MPO-ANCA affinity was assessed by antigen-inhibition ELISAs. The sera from all patients were employed to inhibit biotin-conjugated affinity-purified human anti-MPO antibodies (Probe-biotin), from plasma of a patient with MPA, in a competitive inhibition ELISAs system. The anti-MPO IgG subclass distribution, affinity and inhibition rates to Probe-biotin were evaluated and compared between the WG and MPA groups.Results: Clinical data of patients with WG and MPA were comparable, except for BVAS score. BVAS scores of patients with WG were significantly higher than those with MPA (18.2±6.1 vs. 14.0±4.2, P=0.018). All the patients were MPO-ANCA positive and all four subclasses of anti-MPO IgG could be detected in the fifteen sera from patients with WG. 14/15(93%), 11/15(73%), 11/15(73%) and 13/15(87%) were positive for IgG1, IgG2, IgG3 and IgG4 of MPO-ANCA, respectively. In the twenty-one sera from patients with MPA, 17/21(81%), 14/21 (67%), 14/21(67%) and 16/21(76%) were positive for IgG1, IgG2, IgG3 and IgG4 of MPO-ANCA, respectively. There was no significant difference in titers of IgG1, IgG2 and IgG3 between the two groups. The mean titer of anti-MPO IgG4 subclass in patients with WG was significantly higher than those with MPA (1:1878 vs. 1:218,P<0.005). The median avidity constant of MPO-ANCA was both 35×107M-1 in patients with WG and MPA (0.3-70×10⁷M^-1 vs. 0.3-140×10⁷M^-1, respectively). In WG group, eleven out of the 15 sera could inhibit the binding of Probe-biotin from 32.9% to 69.7%, the average inhibition rate was 47.7%±11.5%. In MPA group, 18/21 sera could inhibit Probe-biotin from 39.2% to 86.6%, the average inhibition rates were 61.7%±14.5%. The average inhibition rate for Probe-biotin in MPA group was significantly higher than that in WG group (P=0.01).Conclusions: MPO-ANCA IgG4 subclass might play a role in the development of WG. The MPO-ANCA in WG and MPA might recognize overlapping but different epitopes on native MPO molecule. The difference in immunological characteristics of MPO-ANCA might contribute to different disease expression.